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- EMDB-3787: Cryo-electron tomography averaged map of microtubule doublet from... -

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Basic information

Entry
Database: EMDB / ID: EMD-3787
TitleCryo-electron tomography averaged map of microtubule doublet from p23 mutant Chlamydomonas axoneme
Map dataSubaverage after subtomogram classification of Chlamydomonas pf23 cilia, based on outer arm dynein structure.
Sample
  • Organelle or cellular component: Ciliary microtubule doublet with dynein, radial spokes and other binding proteins
Biological speciesChlamydomonas reinhardtii (plant)
Methodsubtomogram averaging / cryo EM / Resolution: 50.0 Å
AuthorsSale WS / Ishikawa T / Yamamoto R / Obbineni JM / Alford LM / Hwang J / Ide T / Owa M / Kon T / Inaba K ...Sale WS / Ishikawa T / Yamamoto R / Obbineni JM / Alford LM / Hwang J / Ide T / Owa M / Kon T / Inaba K / Noliyanda J / King SM / Dutcher S
CitationJournal: PLoS Genet / Year: 2017
Title: Chlamydomonas DYX1C1/PF23 is essential for axonemal assembly and proper morphology of inner dynein arms.
Authors: Ryosuke Yamamoto / Jagan M Obbineni / Lea M Alford / Takahiro Ide / Mikito Owa / Juyeon Hwang / Takahide Kon / Kazuo Inaba / Noliyanda James / Stephen M King / Takashi Ishikawa / Winfield S ...Authors: Ryosuke Yamamoto / Jagan M Obbineni / Lea M Alford / Takahiro Ide / Mikito Owa / Juyeon Hwang / Takahide Kon / Kazuo Inaba / Noliyanda James / Stephen M King / Takashi Ishikawa / Winfield S Sale / Susan K Dutcher /
Abstract: Cytoplasmic assembly of ciliary dyneins, a process known as preassembly, requires numerous non-dynein proteins, but the identities and functions of these proteins are not fully elucidated. Here, we ...Cytoplasmic assembly of ciliary dyneins, a process known as preassembly, requires numerous non-dynein proteins, but the identities and functions of these proteins are not fully elucidated. Here, we show that the classical Chlamydomonas motility mutant pf23 is defective in the Chlamydomonas homolog of DYX1C1. The pf23 mutant has a 494 bp deletion in the DYX1C1 gene and expresses a shorter DYX1C1 protein in the cytoplasm. Structural analyses, using cryo-ET, reveal that pf23 axonemes lack most of the inner dynein arms. Spectral counting confirms that DYX1C1 is essential for the assembly of the majority of ciliary inner dynein arms (IDA) as well as a fraction of the outer dynein arms (ODA). A C-terminal truncation of DYX1C1 shows a reduction in a subset of these ciliary IDAs. Sucrose gradients of cytoplasmic extracts show that preassembled ciliary dyneins are reduced compared to wild-type, which suggests an important role in dynein complex stability. The role of PF23/DYX1C1 remains unknown, but we suggest that DYX1C1 could provide a scaffold for macromolecular assembly.
History
DepositionJul 6, 2017-
Header (metadata) releaseAug 16, 2017-
Map releaseSep 6, 2017-
UpdateSep 20, 2017-
Current statusSep 20, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: -0.23
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: -0.23
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3787.png

Downloads & links

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Map

FileDownload / File: emd_3787.map.gz / Format: CCP4 / Size: 47.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubaverage after subtomogram classification of Chlamydomonas pf23 cilia, based on outer arm dynein structure.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
5.68 Å/pix.
x 232 pix.
= 1317.76 Å		5.68 Å/pix.
x 232 pix.
= 1317.76 Å		5.68 Å/pix.
x 232 pix.
= 1317.76 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 5.68 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.001 / Movie #1: -0.23
Minimum - Maximum-0.70259494 - 0.6101861
Average (Standard dev.)0.000158723 (±0.091841586)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions232232232
Spacing232232232
CellA=B=C: 1317.76 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.685.685.68
M x/y/z232232232
origin x/y/z0.0000.0000.000
length x/y/z1317.7601317.7601317.760
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS232232232
D min/max/mean-0.7030.6100.000

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Supplemental data

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Sample components

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Entire : Ciliary microtubule doublet with dynein, radial spokes and other ...

EntireName: Ciliary microtubule doublet with dynein, radial spokes and other binding proteins
Components
  • Organelle or cellular component: Ciliary microtubule doublet with dynein, radial spokes and other binding proteins

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Supramolecule #1: Ciliary microtubule doublet with dynein, radial spokes and other ...

SupramoleculeName: Ciliary microtubule doublet with dynein, radial spokes and other binding proteins
type: organelle_or_cellular_component / ID: 1 / Parent: 0
Source (natural)Organism: Chlamydomonas reinhardtii (plant) / Strain: pf23 / Organelle: cilia

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7.4 / Component - Name: HMDEK
Details: 30 mM HEPES, 5 mM MgSO4, 1 mM DTT, 1 mM EGTA, and 50 mM potassium acetate, pH7.4
GridModel: Plano lacey / Material: COPPER / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 70 % / Chamber temperature: 298 K / Instrument: HOMEMADE PLUNGER / Details: one side blotting.

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Electron microscopy

MicroscopeJEOL 2200FS
Specialist opticsEnergy filter - Name: JEOL in-column / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 25 eV
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 2 / Average exposure time: 1.0 sec. / Average electron dose: 0.8 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal magnification: 20000
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN

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Image processing

Final reconstructionNumber classes used: 1 / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 50.0 Å / Resolution method: OTHER / Software - Name: SPIDER
Details: Visual estimation, compared with datasets in the past
Number subtomograms used: 464
ExtractionNumber tomograms: 5 / Number images used: 660
Final 3D classificationNumber classes: 5 / Avg.num./class: 120 / Software - Name: Xmipp
Details: This particular uploaded map is from 464 subtomograms.
Final angle assignmentType: OTHER / Software - Name: SPIDER / Details: 3D cross correlation by Spider

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